Evaluating the potential for undesired genomic effects of the piggyBac transposon system in human cells
Autor: | Matthew H. Wilson, Sunandan Saha, Cliona M. Rooney, Elizabeth M. Charron, Lauren E. Woodard, Richard C. Welch |
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Rok vydání: | 2015 |
Předmět: |
Transposable element
Genetics Chromosomes Artificial Bacterial Genome Human Green Fluorescent Proteins Transposases Retrotransposon Biology Sleeping Beauty transposon system Polymerase Chain Reaction P element HEK293 Cells Composite transposon PiggyBac Transposon System DNA Transposable Elements Humans Transgenes 5' Untranslated Regions Induced pluripotent stem cell Molecular Biology Transposase DNA Damage Plasmids |
Zdroj: | Nucleic Acids Research |
ISSN: | 1362-4962 0305-1048 |
Popis: | Non-viral transposons have been used successfully for genetic modification of clinically relevant cells including embryonic stem, induced pluripotent stem, hematopoietic stem and primary human T cell types. However, there has been limited evaluation of undesired genomic effects when using transposons for human genome modification. The prevalence of piggyBac(PB)-like terminal repeat (TR) elements in the human genome raises concerns. We evaluated if there were undesired genomic effects of the PB transposon system to modify human cells. Expression of the transposase alone revealed no mobilization of endogenous PB-like sequences in the human genome and no increase in DNA double-strand breaks. The use of PB in a plasmid containing both transposase and transposon greatly increased the probability of transposase integration; however, using transposon and transposase from separate vectors circumvented this. Placing a eGFP transgene within transposon vector backbone allowed isolation of cells free from vector backbone DNA. We confirmed observable directional promoter activity within the 5′TR element of PB but found no significant enhancer effects from the transposon DNA sequence. Long-term culture of primary human cells modified with eGFP-transposons revealed no selective growth advantage of transposon-harboring cells. PB represents a promising vector system for genetic modification of human cells with limited undesired genomic effects. |
Databáze: | OpenAIRE |
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